Experimental Study On The Detection Of Chlamydia By Ring - Mediated Isothermal Amplification

Objective: To develop the LAMP for detection of Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci respectively, for the grass-roots and in-situ detection of rapid and sensitive detection method of practical molecular biology.Methods: Using Giemsa staining or immunofluorescence staining to identify 3 types of chlamydia in cell cultures. Four or six primers were designed to amplificate target gene of Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci, respectively. Bacterial genomic DNA was extracted by Kit. Through optimizing the amplification conditions, we have established specifical LAMP for the correspondence pathogens. Target DNA amplified under isothermal conditions(60~65°C) for 60 min, then, results were judged by naked eyes and electrophoretic analysis. The products of LAMP were confirmed by digestion using restriction enzymes. To evaluate the specificity of the LAMP assay, other pathogens were used as controls and detected by LAMP. To compare the sensitivity between LAMP and PCR, target pathogens DNA were 10-fold serially diluted and were amplified by LAMP and PCR, respectively.Results: The three species of Chlamydia-specific inclusions can be observed by the Giemsa staining or immunofluorescence staining. After LAMP reaction, ladder patterns unique to the LAMP assay were observed with four LAMP for the target pathogens. Both naked eyes and electrophoretic analysis were able to detect the products in the LAMP assay. Amplification was not observed when negative controls were tested. The specificity of LAMP products for the target pathogens were also confirmed by restriction enzymes digestion. The result demonstrate that LAMP with equal sensitivity to conventional PCR assay, and the detection limit of LAMP assay for Ct, Cpn and Cps DNA was 1 pg/μL, 100 pg/μL, 0.1 pg/μL, respectively.Conclusion: LAMP assay for detection bacterial simple Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci have been established and the assay is rapid, specific and sensitive.